JPWO2011055737A1 - Thermostable strand displacement DNA polymerase and method for producing the DNA polymerase - Google Patents

Abstract

The present invention provides a novel heat-resistant strand displacement DNA polymerase and a method for producing the same. A strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3. A strand displacement type DNA polymerase comprising a strand displacement type DNA polymerase activity comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2. The temperature at which the strand displacement DNA polymerase activity by the RCA method is maximized is in the range of 60 to 75 ° C., or has the strand displacement DNA polymerase activity by the LAMP method at a temperature of 70 ° C., or 70 to 80 ° C. The strand displacement DNA polymerase activity after heat treatment for 5 minutes at a temperature has a heat resistance of 80% or more of the strand displacement DNA polymerase activity by the LAMP method when not heat-treated. A strand displacement type DNA polymerase comprising a strand displacement type DNA polymerase activity, comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3. The temperature at which the strand displacement DNA polymerase activity by the RCA method is maximized is in the range of 45 to 55 ° C., has the strand displacement DNA polymerase activity by the LAMP method at a temperature of 55 ° C., or a temperature of 55 to 60 ° C. The strand displacement type DNA polymerase activity after heat treatment for 5 minutes has a heat resistance of 80% or more of the strand displacement type DNA polymerase activity by the LAMP method when not heat-treated.

Description

Cross-reference of related applications

  This application claims the priority of Japanese Patent Application No. 2009-254529 filed on Nov. 6, 2009, the entire description of which is specifically incorporated herein by reference.

  The present invention relates to a thermostable strand displacement type DNA polymerase and a method for producing the DNA polymerase.

  In recent years, strand displacement type DNA amplification such as SDA (Strand Displacement Amplification) method and LAMP method has been attracting attention and frequently used as a DNA amplification method.

  In order to perform strand displacement type DNA amplification, a DNA polymerase having strand displacement type activity is used. As DNA polymerase having strand displacement activity, for example, Bst DNA polymerase derived from Geobacillus stearothermophilus, Bpa DNA polymerase derived from Bacillus pallidus [Patent Document 1] and the like have been reported. In addition, as in E. coli, a Klenow-type enzyme that does not contain a 5′-3 ′ exonuclease domain on the N-terminal side of a protein is also known [Patent Document 2]. A DNA polymerase derived from the genus Bacillus grows in a moderate high temperature range (a temperature around 60 ° C.), has heat resistance and has a strand displacement type DNA polymerase activity.

  Although it does not have heat resistance, the DNA polymerase of Phi29 phage that infects Bacillus subtilis has a strong 3'-5 'exonuclease activity as well as T4 DNA polymerase, and has a strand displacement type DNA polymerase activity. . Recently, these strand displacement DNA polymerases have been used for sample preparation and base sequence decoding in large-scale sequencing using an ultrafast parallel DNA sequencer, and their usefulness is high.

US Patent 5,736,373 US Patent 5,814,506 Japanese Patent No.4086783 Japanese Patent No. 4086235 Japanese Unexamined Patent Publication No. 2001-321181

Sidhu, M. K., et al. BioTechniques., 21: 44 (1998) Kiefer, J. R., et al. Structure., 5: 95 (1997) Sommer, R., Tautz, D. Nucl. Acids. Res., 17: 6749 (1989) Collins, F. S., and Weissman, S. M. Proc. Natl. Acad. Sci. U S A., 81: 6812 (1984) Triglia, T., Peterson, M. G., and Kemp, D. J. Nucl. Acids. Res., 16: 8186 (1988) Xu, S. Y., Xiao, J. P., Posfai, J., Maunus, R. E., Benner, J. S. Nucl. Acids. Res., 25: 3991 (1988)

  The entire descriptions of Patent Documents 1 to 5 and Non-Patent Documents 1 to 6 are specifically incorporated herein by reference.

  Among DNA sequences having a high GC content, there are DNA sequences that are extremely difficult to amplify by PCR. In such a case, there is a method in which amplification is possible by adding a solvent that affects hydrogen bonding (for example, DMSO, betaine, formamide) [for example, Non-Patent Document 1]. However, in strand displacement type DNA amplification, amplification is easy even for a DNA sequence having a high GC content as described above. Therefore, strand displacement type DNA amplification is a promising technique in the field of gene analysis, testing and diagnosis in the future.

  However, the strand displacement type DNA polymerases reported so far have lower heat resistance than Taq DNA polymerase, Pfu DNA polymerase, etc. which are widely used in PCR. In order to obtain accurate results in genetic analysis, testing, and diagnosis, there is a demand for DNA polymerases having new functions such as better protein stability and higher resistance to inhibitors.

  Accordingly, an object of the present invention is to provide a novel strand-displacement DNA polymerase having heat resistance and a production method thereof.

  In order to achieve the above object, the present inventors investigated and examined DNA polymerase derived from microorganisms present in compost obtained by fermenting organic waste at a temperature of 85 ° C. or higher. The present invention has been completed by finding a novel thermostable DNA polymerase having heat resistance that achieves the above.

The present invention is as follows.
[1]
A strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2.
[2]
A temperature comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, having a strand displacement type DNA polymerase activity, and having a maximum strand displacement type DNA polymerase activity by the RCA method. A strand displacement DNA polymerase in the range of 60-75 ° C.
[3]
It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has a strand displacement type DNA polymerase activity, and has a strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 70 ° C. A strand displacement DNA polymerase.
[4]
A strand displacement type comprising an amino acid sequence having 90% or more identity with the amino acid sequence described in SEQ ID NO: 1 or 2, having strand displacement type DNA polymerase activity, and after heat treatment at a temperature of 70 to 80 ° C. for 5 minutes A strand displacement type DNA polymerase having a heat resistance of 80% or more of the strand displacement type DNA polymerase activity by the LAMP method when the DNA polymerase activity is not heat-treated.
[5]
The strand displacement DNA polymerase according to [4], wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 60 to 75 ° C.
[6]
The strand displacement DNA polymerase according to [4], which has strand displacement DNA polymerase activity according to the LAMP method at a temperature of 70 ° C.
[7]
The strand displacement DNA polymerase according to any one of [2] to [6], wherein the pH at which the strand displacement DNA polymerase activity is maximized is 8.0 or more.
[8]
Any one of [2] to [7], wherein the amino acid sequence shown in SEQ ID NO: 1 or 2 contains at least one or more biologically acceptable amino acid substitutions, amino acid deletions, and / or amino acid insertions The strand displacement type DNA polymerase as described.
[9]
The strand displacement type DNA polymerase according to [8], comprising 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.
[10]
A strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 3.
[11]
It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has a strand displacement type DNA polymerase activity, and has a temperature at which the strand displacement type DNA polymerase activity by the RCA method becomes maximum at 45 to 45%. Strand displacement DNA polymerase in the range of 55 ° C.
[12]
It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has strand displacement type DNA polymerase activity, and has strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 55 ° C. Strand displacement DNA polymerase.
[13]
A strand-displacement DNA polymerase comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement-type DNA polymerase activity, and subjected to heat treatment at a temperature of 55-60 ° C for 5 minutes A strand displacement DNA polymerase having a heat resistance of 80% or more of the strand displacement DNA polymerase activity by the LAMP method when the activity is not heat-treated.
[14]
The strand displacement DNA polymerase according to [13], wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 45 to 55 ° C.
[15]
The strand displacement DNA polymerase according to [13], which has strand displacement DNA polymerase activity according to the LAMP method at a temperature of 55 ° C.
[16]
The strand displacement type DNA polymerase according to any one of [11] to [15], wherein the pH at which the DNA polymerase activity and the strand displacement type activity are maximized is 8.0 or more.
[17]
The amino acid sequence shown in SEQ ID NO: 3 contains at least one or more biologically acceptable amino acid substitutions, amino acid deletions, and / or amino acid insertions, according to any one of [11] to [16] Strand displacement DNA polymerase.
[18]
The strand displacement DNA polymerase according to [17], comprising 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.
[19]
The strand displacement DNA polymerase according to any one of [1] to [18], which is used for nucleic acid synthesis in a LAMP (Loop-mediated isothermal amplification) method.
[20]
[1] A gene encoding the DNA polymerase according to any one of [18].
[21]
[20] The gene described in [20] is mounted on a vector, and after the host cell is transformed with this vector, the transformed host cell is cultured, and the DNA polymerase encoded by the gene is accumulated in the culture, A method for producing a strand displacement type DNA polymerase, comprising collecting accumulated DNA polymerase.
[22]
The production method according to [20], wherein the collected DNA polymerase comprises the strand displacement type DNA polymerase according to any one of [1] to [18].
[23]
The production method according to [20] or [21], wherein an E. coli expression vector using a co-expression method with a leader sequence is used.

  According to the present invention, it is an object of the present invention to provide a novel strand-displacement DNA polymerase having heat resistance and a production method thereof. Furthermore, the strand displacement type DNA polymerase of the present invention has a good strand displacement type DNA polymerase even under basic conditions, and also has excellent storage stability of the strand displacement type DNA polymerase activity.

FIG. 3 is an explanatory view showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (fYMO81 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). FIG. 3 is an explanatory view showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (fYMO81 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). FIG. 3 is an explanatory view showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (tYMO81 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). FIG. 3 is an explanatory view showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (tYMO81 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). It is explanatory drawing which shows the comparison of the amino acid sequence of DNA polymerase (GenBank Accession No. AAB52611) derived from thermostable DNA polymerase (t96-7 DNA polymerase) derived from unidentified microorganism 96-7 and Geobacillus stearothermophilus. It is explanatory drawing which shows the comparison of the amino acid sequence of DNA polymerase (GenBank Accession No. AAB52611) derived from thermostable DNA polymerase (t96-7 DNA polymerase) derived from unidentified microorganism 96-7 and Geobacillus stearothermophilus. It is a gel electrophoresis photograph showing the expression of fYMO81 DNA polymerase and tYMO81 DNA polymerase in E. coli. It is a gel electrophoresis photograph showing the expression of t96-7 DNA polymerase in E. coli. 2 is a gel electrophoresis photograph showing the DNA polymerase activity of fYMO81 DNA polymerase and tYMO81 DNA polymerase. It is a gel electrophoresis photograph which shows the DNA polymerase activity of t96-7 DNA polymerase. 2 is a gel electrophoresis photograph showing the optimum reaction temperature in the RCA method for fYMO81 DNA polymerase and tYMO81 DNA polymerase, and a gel electrophoresis photograph showing the optimum reaction temperature in the RCA method for t96-7 DNA polymerase.

[Strand displacement DNA polymerase]
The present invention relates to a strand displacement type DNA polymerase, a strand displacement type DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2 (the strand displacement type DNA polymerase according to the first aspect of the present invention) and SEQ ID NO: 3 The present invention relates to a strand displacement type DNA polymerase comprising the described amino acid sequence (the strand displacement type DNA polymerase according to the second aspect of the present invention).

  As described above, as a result of investigating and examining the DNA polymerase derived from microorganisms present in compost obtained by fermenting organic waste at a temperature of 85 ° C. or higher, the present inventors have found that the first of the present invention described above. The strand-displacing DNA polymerase of the embodiment and the second embodiment was found.

  Geobacillus stearothermophilus that grows at moderately high temperatures is common as a thermophilic aerobic bacterium, but its classification is very ambiguous, and several DNA polymerases derived from Geobacillus stearothermophilus have been reported so far [Non-Patent Document 2]. ]. In addition, DNA polymerases having the same structure as those of the same Geobacillus genus but with the reported stealothermophilus and caldotenax have been discovered. This indicates that the genus Geobacillus, which is a moderately thermophilic bacterium, still has a wide variety, and is an attractive microorganism for exploring new DNA polymerases.

  Mesophilic aerobic spore bacteria belonging to the genus Bacillus and Geobacillus have been used for the production of compost. Compost is a fertilizer obtained by fermenting organic matter such as livestock excrement, manure, sludge, and municipal waste using microorganisms. Conventionally, thermophilic microorganisms have been used in the fermentation process. However, the upper limit of the fermentation temperature by heat of fermentation is about 80 ° C., and spore-forming germs could not be completely removed.

  To solve this problem, bacterial cultures grown at a temperature of 85 ° C or higher obtained from soil in the Kirishima volcanic area are mixed with raw sludge, mixed, aerated and fermented in the sludge at a fermentation temperature of 85 ° C or higher. A method has been found for killing various germs, seeds, etc. present in the plant, purifying sludge, and obtaining a fermented sludge containing only a large number of useful cells [Patent Documents 3 and 4].

  This sludge fermented product is used as compost and has a low occupation rate compared to spore-forming bacteria in conventional compost, and the new mesophilic aerobic spore bacteria belonging to the genus Bacillus and Geobacillus that would have been overlooked Many thermophilic aerobic spore bacteria and thermophilic bacteria have been found [Patent Documents 3 and 4].

  The present inventors pay attention to the above-mentioned compost obtained by fermenting organic waste at a temperature of 85 ° C. or higher, and the strand-substituted DNA polymerase having the heat resistance aimed by the present invention can be obtained from this compost. I tried to find out. As a result, a DNA polymerase gene derived from the thermophilic bacterium Caldothrix satsumae YMO81 (FERM BP-8233) isolated from the compost was isolated. The aforementioned Geobacillus stearothermophilus is a gram-positive gonococcus having a spore-forming ability, but Caldothrix satsumae YMO81 is a gram-negative bacterium and does not have a spore-forming ability. Regarding the obtained DNA polymerase derived from Caldothrix satsumae YMO81, no nucleotide sequence or amino acid sequence was reported. Therefore, the recombinant protein was expressed in Escherichia coli to verify its activity, and the chain according to the first aspect of the present invention was used. It led to substitutional DNA polymerase.

  Furthermore, the DNA polymerase gene was also isolated directly from the metagenome of the microbial community present in the compost. The microorganism obtained here was named 96-7, and for the DNA polymerase derived from 96-7, there was no report of the base sequence or amino acid sequence, so the recombinant protein was expressed in E. coli and the activity was verified. The strand-displacing DNA polymerase according to the second aspect of the present invention has been reached. Although the classification of the microorganism 96-7 has not yet been identified, the DNA polymerase derived from 96-7 has no report of nucleotide sequence or amino acid sequence, and the DNA polymerase derived from 96-7 is also a novel DNA polymerase.

  The strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 1 is composed of 890 amino acids as shown in Examples 1 to 3 and Test Example 1, and the strand displacement DNA polymerase activity by the RCA method is maximized. The temperature is in the range of 60 to 75 ° C., and has the strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 70 ° C., and the strand displacement type DNA polymerase activity after the heat treatment at 70 to 80 ° C. for 5 minutes. It has a heat resistance of 80% or more of the strand displacement type DNA polymerase activity when not heat-treated, and the pH at which the strand displacement type DNA polymerase activity is maximized is 8.0 or more.

  The strand displacement type DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 2 is composed of 608 amino acids as shown in Examples 1 to 3 and Test Example 1, and is composed of the amino acid sequence shown in SEQ ID NO: 1. Similar to the DNA polymerase, the temperature at which the strand displacement DNA polymerase activity by the RCA method is maximized is in the range of 60 to 75 ° C., and has the strand displacement DNA polymerase activity by the LAMP method even at a temperature of 70 ° C. The strand displacement DNA polymerase activity after heat treatment for 5 minutes at a temperature of -80 ° C has a heat resistance of 80% or more of the strand displacement DNA polymerase activity when not heat-treated, and the strand displacement DNA polymerase activity is the maximum The pH is 8.0 or more.

The strand displacement DNA polymerase according to the first aspect of the present invention includes the following strand displacement DNA polymerase in addition to the strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.
(1) It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has strand displacement DNA polymerase activity, and has maximum strand displacement DNA polymerase activity by the RCA method A strand displacement DNA polymerase having a temperature in the range of 60 to 75 ° C. The temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 60 to 70 ° C.
(2) An amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has a strand displacement type DNA polymerase activity, and is a strand displacement type by LAMP method even at a temperature of 70 ° C. A strand displacement DNA polymerase having DNA polymerase activity. It preferably has strand displacement DNA polymerase activity by the LAMP method even at a temperature of 75 ° C., more preferably at a temperature of 80 ° C.
(3) It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has strand displacement type DNA polymerase activity, and is subjected to heat treatment at a temperature of 70 to 80 ° C. for 5 minutes A strand displacement DNA polymerase having a heat resistance of 80% or more of the strand displacement DNA polymerase activity by the LAMP method when the strand displacement DNA polymerase activity is not heat-treated. Preferably, it has a heat resistance of 90% or more, more preferably 95% or more of the strand displacement type DNA polymerase activity by the LAMP method when not heat-treated.
(4) The strand displacement DNA polymerase according to (3), wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 60 to 75 ° C. The temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 60 to 70 ° C.
(5) The strand displacement DNA polymerase according to (3), which has strand displacement DNA polymerase activity according to the LAMP method at a temperature of 70 ° C. It preferably has strand displacement DNA polymerase activity by the LAMP method even at a temperature of 75 ° C., more preferably at a temperature of 80 ° C.
(6) The strand displacement type DNA polymerase according to any one of (1) to (5), which has a high basic activity with a pH of 8.0 or more at which the strand displacement DNA polymerase activity is maximized. Preferably, the strand displacement type DNA polymerase activity is maximized in the pH range of 8.0 to 10.0.
(7) In the strand displacement type DNA polymerase of (1) to (6) above, the amino acid sequence shown in SEQ ID NO: 1 or 2 is at least one biologically acceptable amino acid substitution or amino acid deletion , And / or amino acid insertions, preferably 1-10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.

  In the above (1) to (7), the amino acid sequence shown in SEQ ID NO: 1 or 2 is preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, Preferably, the amino acid sequence having an identity of 99% or more has high activity at high temperature of the strand displacement DNA polymerase, high heat resistance of the strand displacement DNA polymerase activity, and high basic activity. Appropriate from the viewpoint.

The strand displacement DNA polymerase according to the second aspect of the present invention includes the following strand displacement DNA polymerase in addition to the strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 3.
(8) A temperature consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement DNA polymerase activity, and maximizing strand displacement DNA polymerase activity by the RCA method Is a strand displacement DNA polymerase having a temperature in the range of 45-55 ° C. The temperature at which the strand displacement type DNA polymerase activity by the RCA method is maximized is preferably in the range of 50 to 55 ° C.
(9) An amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has strand displacement DNA polymerase activity, and strand displacement DNA polymerase activity by the LAMP method at a temperature of 60 ° C A strand displacement DNA polymerase.
(10) A strand displacement comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement type DNA polymerase activity, and after heat treatment at a temperature of 55-60 ° C for 5 minutes A strand displacement type DNA polymerase having a heat resistance of 80% or more of the strand displacement type DNA polymerase activity by the LAMP method when the type DNA polymerase activity is not heat-treated. Preferably, it has a heat resistance of 90% or more, more preferably 95% or more of the strand displacement type DNA polymerase activity by the LAMP method when not heat-treated.
(11) The strand displacement DNA polymerase according to (10), wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 45 to 55 ° C. The temperature at which the strand displacement type DNA polymerase activity by the RCA method is maximized is preferably in the range of 50 to 55 ° C.
(12) The strand displacement DNA polymerase according to (10), which has strand displacement DNA polymerase activity by a LAMP method at a temperature of 55 ° C. Preferably, it has strand displacement DNA polymerase activity by the LAMP method even at a temperature of 60 ° C.
(13) The strand displacement type DNA polymerase according to any one of (8) to (12), which has a high basic activity with a pH of 8.0 or more at which the strand displacement DNA polymerase activity is maximized. Preferably, the strand displacement DNA polymerase activity is maximized when the pH is in the range of 8.0 to 9.0.
(14) In the strand displacement type DNA polymerase of (8) to (13) above, the amino acid sequence shown in SEQ ID NO: 3 is at least one biologically acceptable amino acid substitution, amino acid deletion, and An amino acid insertion can be included, and preferably includes 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.

  In the above (8) to (14), it is preferably 95% or more, more preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, most preferably the amino acid sequence shown in SEQ ID NO: 3. From the viewpoint of comprising an amino acid sequence having 99% or more identity, the strand displacement type DNA polymerase has high activity at high temperature, the heat resistance of the strand displacement type DNA polymerase activity is high, and the basic activity is also high. Is appropriate.

  The strand displacement type DNA polymerase of the present invention can be used for various nucleic acid synthesis to which the strand displacement type DNA polymerase activity is applied, and is preferably used for nucleic acid synthesis in a LAMP (Loop-mediated isothermal amplification) method.

  The present invention includes a gene encoding the strand displacement DNA polymerase of the present invention. Representative examples of the gene of the present invention are shown in SEQ ID NOs: 4-6.

  The method for obtaining the strand displacement type DNA polymerase of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When producing a recombinant protein, a gene (DNA) encoding the protein is obtained as described later. By introducing this DNA into an appropriate expression system, the protein of the present invention (strand displacement DNA polymerase) can be produced.

  The strand-displacing DNA polymerase of the present invention has a gene encoding the strand-displacing DNA polymerase of the present invention mounted on a vector, transforms the host cell with this vector, and then cultures the transformed host cell. The DNA polymerase encoded by the gene can be accumulated in the culture, and the accumulated DNA polymerase can be collected by a production method.

  The method for obtaining a gene encoding the strand displacement DNA polymerase of the present invention is not particularly limited. The gene encoding the strand-displacing DNA polymerase of the present invention can be obtained by, for example, chemical synthesis or genetic engineering techniques based on the amino acid sequence described in SEQ ID NOs: 1 to 3 and the base sequence information described in SEQ ID NOs: 4-6. Alternatively, it can be produced by any method known to those skilled in the art, such as mutagenesis.

For example, it can be carried out by using a method of contacting a DNA having the base sequence described in SEQ ID NOs: 4 to 6 with a drug as a mutagen, a method of irradiating with ultraviolet rays, a genetic engineering method, etc. . Site-directed mutagenesis method which is one of genetic engineering technique is useful because it is a method capable of introducing a specific mutation into a specific position, Molecular Cloning:. A laboratory Mannual , 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter, Current Protocols (Abbreviated as in-molecular biology) and the like.

  Appropriate probes and primers are prepared based on the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing in this specification or the information of the base sequence shown in SEQ ID NO: 4 or 5, and using them, Caldothrix satsumae YMO81 The gene of the present invention can be isolated by screening a cDNA library. A cDNA library can be prepared from Caldothrix satsumae YMO81 by a conventional method.

  A gene encoding the strand displacement type DNA polymerase of the present invention can also be obtained by PCR. Using the above Caldothrix satsumae YMO81 cDNA library as a template, PCR is performed using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 4 or 5. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  The above-described procedures such as probe or primer preparation, cDNA library construction, cDNA library screening, and target gene cloning are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols In -It can be performed according to the method described in Molecular Biology etc.

  The gene of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes. Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

Promoters that can operate in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, and the Bacillus amyloliquefati. Enns-BAN amylase gene (Bacillus amyloliquefaciens BAN amylase gene), Bacillus subtilis alkaline protease gene (Bacillus subtilis alkaline protease gene) or promoters of the Bacillus pumilus-xylosidase gene (Bacillus pumilus xylosldase gene) or phage lambda, P _R or P _L promoters, lac of E.coli, such as trp or tac promoter and the like.

  Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are promoters. Examples of a promoter operable in a yeast host cell include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, an ADH2-4c promoter, and the like. Examples of promoters that can operate in filamentous fungal cells include the ADH3 promoter or the tpiA promoter.

  Moreover, the gene of the present invention may be operably linked to an appropriate terminator as necessary. The recombinant vector containing the gene of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like. The recombinant vector containing the gene of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). Is mentioned.

  The recombinant vector containing the gene of the present invention may further contain a selection marker. Selectable markers include, for example, genes whose complement is lacking in host cells such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI gene, or ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin. Methods for ligating the gene, promoter, and optionally terminator and / or secretory signal sequence of the present invention and inserting them into an appropriate vector are well known to those skilled in the art.

  A transformant can be prepared by introducing a recombinant vector containing the gene of the present invention into an appropriate host. The host cell into which the recombinant vector containing the gene of the present invention is introduced may be any cell as long as it can express the gene of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.

  Examples of bacterial cells include Gram positive bacteria such as Bacillus or Streptomyces or Gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method. Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

  Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevis 1ae or Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  Examples of other fungal cells are cells belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).

As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.
Insect cells include Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York, Spodoptera frugiperda ovarian cells. (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.
Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include the calcium phosphate method and the lipofection method.

  The transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to isolate and purify the protein of the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used. For example, when the protein of the present invention is expressed in a dissolved state in the cell, after the culture is completed, the cell is collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Using the hydrophobic chromatography method used, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination, the strand displacement of the present invention Type DNA polymerase can be obtained as a purified preparation.

  E. coli recombinant protein expression vector can be used for expression of the above recombinant protein. When using E. coli recombinant protein expression vector, Expression Vector pLEAD5 DNA (manufactured by Nippon Gene) using co-expression with leader sequence [ The use of Patent Document 5] is preferable from the viewpoint of a vector optimized for expressing a heterologous gene with a biased GC or AT content in E. coli. In general, genes with high GC content derived from thermophiles and actinomycetes are often difficult to express in E. coli, and the cause is thought to be due to low translation efficiency. Expression Vector pLEAD5 is constructed based on the results of screening an expression vector with high protein expression efficiency from a gene with a high GC content, and should be effective for the expression of genes with a high AT content. It is shown.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1
[Cloning of DNA polymerase I from Caldothrix satsumae YMO81]
In DNA polymerase I, a degenerate primer is designed from the amino acid sequence of a DNA polymerase domain that is highly conserved among different organisms, and a gene is obtained by PCR using the already obtained Caldothrix satsumae YMO81 genomic DNA as a template DNA. [Non-patent Document 3 (Sommer, R., Tautz, D. Nucleic Acids Research, 17: 6749 (1989))]. The sequence of the obtained DNA fragment was analyzed by the Sanger method, and homology with DNA polymerase I was confirmed.

  Next, Caldothrix satsumae YMO81 genomic DNA was digested with an arbitrary restriction enzyme, and the DNA fragment was circularized by self-ligation. The sequence before and after the obtained DNA fragment was sequentially isolated by the inverse PCR method using this as a template DNA [Non-patent Document 4 (Collins, FS, and Weissman, SM Proc Natl Acad Sci USA. 81: 6812 (1984 )), Non-Patent Document 5 (Triglia, T., Peterson, MG, and Kemp, DJ Nucleic Acids Reseach, 16: 8186 (1988)), Non-Patent Document 6 (Xu, SY, Xiao, JP, Posfai, J.). , Maunus, RE, Benner, JS Nucleic Acids Research, 25: 3991 (1988))]. By repeating this inverse PCR method, the full length of the DNA polymerase I gene was isolated and sequenced.

  The predicted sequence of the isolated DNA polymerase I gene is 2,673 bp, and is estimated to encode a protein composed of 890 amino acids. A comparison between the obtained amino acid sequence and the amino acid sequence (SEQ ID NO: 23) of Geobacillus stearothermophilus-derived DNA polymerase I (GenBank Accession No. AAB52611) is shown in FIG. 1, and the domain presumed to be a 5′-3 ′ exonuclease domain FIG. 2 shows a comparison between the amino acid sequence obtained by removing E. coli and the amino acid sequence (SEQ ID NO: 23) of DNA polymerase I (GenBank Accession No. AAB52611) derived from Geobacillus stearothermophilus.

  In comparison of deduced amino acid sequences, the homology at the amino acid level between DNA polymerase I derived from Caldothrix satsumae YMO81 and DNA polymerase I derived from DNA Geobacillus stearothermophilus was 58%.

Example 2
[Construction of a DNA polymerase expression vector derived from Caldothrix satsumae YMO81]
PCR primer (fYMO81_N: 5'-GTG ATG GAG AAG CTG GTC) for introducing a 890 amino acid residue, 2,670 bp gene corresponding to the full length of the DNA polymerase gene from Caldothrix satsumae YMO81 into the protein expression vector Expression Vector pLEAD5 DNA CT-3 ', tYMO81_C_SacI: 5'-GCT GAG CTC TAT TTC GCG TCG TAC CAC-3') was designed. The base sequences of the designed primers (fYMO81_N, tYMO81_C_SacI) are shown in SEQ ID NOs: 7 and 8.

  FIG. 4 is an acrylamide gel electrophoresis photograph showing expression in Escherichia coli of a DNA polymerase derived from Caldothrix satsumae YMO81. In FIG. 4, Lane 2 confirms the expression of the recombinant protein from the full length gene of the cloned DNA polymerase. First, after heat treatment at 94 ° C for 2 minutes, the target DNA fragment is obtained by PCR method, which is 35 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds and 68 ° C for 3 minutes. Amplified. Since a region that is difficult to amplify by PCR was included, a DNA fragment was obtained by PCR in the presence of 5% formamide. The 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, and one end was further digested with SacI. Then, only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene). When E. coli JM109 was transformed with this DNA and the expression of the protein in the transformant was confirmed, it was confirmed that a recombinant protein having an expected molecular weight of about 113 kDa was expressed (FIG. 4, lane 2). ). The sequence of 890 amino acids encoding this protein is shown in SEQ ID NO: 1 described later.

  Lane 5 confirms the expression of DNA polymerase from which the 5′-3 ′ exonuclease domain has been removed. That is, to remove the 5'-3 'exonuclease domain, the PCR primer (tYMO81_N: 5'-GTG) was used to introduce a gene of 608 amino acid residues and 1,824 bp in which the 284th amino acid was replaced by methionine. ATG CCC CAT GTG CCG-3 ', tYMO81_C_SacI) was designed, heat-treated at 94 ° C for 2 minutes, then totaled for 94 seconds for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 2 minutes The DNA fragment of interest was amplified by PCR using 35 cycles. The 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, and one end was further digested with SacI. Then, only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene). The base sequence of the designed primer (tYMO81_N) is shown in SEQ ID NO: 9. When E. coli JM109 was transformed with this DNA and the expression of the transformant was confirmed, it was confirmed that a recombinant protein having an expected molecular weight of about 77 kDa was expressed (FIG. 4, lane 5). The sequence of 608 amino acids encoding this protein is shown in SEQ ID NO: 2 described later.

  These proteins of about 113 kDa (referred to as fYMO81 DNA polymerase) and about 77 kDa (referred to as tYMO81 DNA polymerase) were purified, and the DNA polymerase activity was examined by the RCA (Rolling Circle Amplification) method. A circular single-stranded DNA derived from Escherichia coli phage was used as a template, and primers were added to this DNA to verify the presence or absence of DNA polymerase activity and strand displacement activity. As the enzyme control, 5'-3 'exonuclease-deficient DNA polymerase derived from Geobacillus stearothermophilus was used (Bst DNA Polymerase, Large Fragment; New England Biolabs).

Specifically, M13mp18 single strand DNA 20 ng, Universal primer (5'-GTT TTC CCA GTC ACG ACG TTG TA-3 ') 50 nM, 0.25 mM each dNTPs, 20 mM Tris-HCl (pH 8.8 at 25 ° C) , 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Tween 20, and DNA polymerase 20 μl in total were reacted at 60 ° C for 0 to 30 minutes, followed by agarose gel electrophoresis As a result, the DNA band shifted to the polymer side in a reaction time-dependent manner, suggesting that fYMO81 DNA polymerase and tYMO81 DNA polymerase have a strand displacement type DNA polymerase activity (FIG. 6). . The nucleotide sequence of Universal primer is shown in SEQ ID NO: 10.

Example 3
[Purification of recombinant DNA polymerase from Caldothrix satsumae YMO81]
The E. coli obtained in Example 2 is cultured and collected. E. coli is suspended in Sonication Buffer (200 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol, 0.1 mM Benzamidine, 10 μg / ml Bacitracin, 30 μg / ml Phenylmethylsulfonyl fluoride, 20 mM Tris-HCl (pH 7.5 at 25 ° C.)) Decompose genomic DNA by sonication while lowering the temperature on ice. The suspension is heated in a 55 ° C. water bath for 20 minutes. Immediately lower the temperature on ice, centrifuge and collect the supernatant.

  The supernatant was added to an anion exchange column (HiTrap Q HP; manufactured by GE Healthcare Bioscience), washed thoroughly, and separated by a linear NaCl gradient.

  RCA method and SDS-PAGE were performed to confirm fractions containing DNA polymerase. The desired fractions were collected, added to a heparin affinity column (HiTrap Heparin HP; manufactured by GE Healthcare Bioscience), washed thoroughly, and then separated by a NaCl linear concentration gradient.

  SDS-PAGE was performed to confirm fractions containing DNA polymerase (FIG. 4, lane 3 and lane 6). Collected fractions were tested using well dialyzed against storage buffer (0.1 mM EDTA, 1 mM Dithiothreitol, 10 mM Tris-HCl (pH 7.5 at 25 ° C), 0.1% Tween 20, 50% Glycerol). The analysis in Example 1 was performed.

Example 4
[Cloning of 96-7-derived DNA polymerase I]
In general, many organisms such as bacteria are symbiotic in nature, and pure culture of specific bacteria is often difficult. Therefore, the present inventors tried a method of extracting metagenomic DNA from composting during fermentation without isolating bacteria and isolating DNA polymerase I gene by PCR.

  First, the present inventors include a bacterial culture grown at a temperature of 85 ° C. or higher obtained from soil in the Kirishima volcanic area, and a high temperature portion around 90 ° C. from compost during fermentation using sewage sludge as a main raw material. The DNA was extracted using QIAamp DNA Stool Mini Kit (Qiagen Co., Ltd.).

  In DNA polymerase I, a degenerate primer was designed from the amino acid sequence of a DNA polymerase domain that is particularly highly conserved among different organisms, and a part of the gene was obtained by PCR using the obtained metagenomic DNA as a template DNA. The sequence of the obtained DNA fragment was analyzed by the Sanger method, and homology with DNA polymerase I was confirmed. The microorganism presumed to have this DNA fragment in the genomic DNA was designated as 96-7. The classification of this microorganism 96-7 has not been identified yet.

  Next, the metagenomic DNA was digested with an arbitrary restriction enzyme, and the DNA fragment was circularized by self-ligation. The sequence before and after the obtained DNA fragment was sequentially isolated by the inverse PCR method using this as a template DNA. By repeating this inverse PCR method, the sequence of the region excluding the sequence presumed to encode the 5′-3 ′ exonuclease domain of the DNA polymerase I gene was determined.

  The predicted sequence for the isolated DNA polymerase I gene is 1,785 bp, and was predicted to encode a protein composed of 594 amino acids. A comparison between the obtained amino acid sequence and the amino acid sequence (SEQ ID NO: 23) of DNA polymerase I (GenBank Accession No. AAB52611) derived from Geobacillus stearothermophilus is shown in FIG.

  In comparison of the deduced amino acid sequences, the homology at the amino acid level of the DNA polymerase I derived from Geobacillus stearothermophilus with the 5'-3 'exonuclease domain of 96-7 derived DNA polymerase I and the region not including the deduced domain The sex was 66%.

Example 5
[Construction of DNA polymerase expression vector derived from 96-7]
PCR primers (t967_N: 5'-GTG ATG TCC GGA CAA AAG GAT G-3 ', t967_C_SacI: 5'-GAG GAG CTC TAT to introduce the DNA polymerase gene derived from 96-7 into the protein expression vector Expression Vector pLEAD5 DNA TTT GCA TCG AAC CAG-3 ') was designed. The nucleotide sequences of the designed primers (t967_N, t967_C_SacI) are shown in SEQ ID NOs: 11 and 12, respectively.

  FIG. 5 is an acrylamide gel electrophoresis photograph showing the expression of 96-7-derived DNA polymerase in E. coli. In FIG. 5, lane 2 confirms the expression of DNA polymerase from which the 5′-3 ′ exonuclease domain has been removed. That is, in order to remove the 5'-3 'exonuclease domain, the above-mentioned primer (t967_N, t967_C_SacI) was used to introduce a 595 amino acid residue, 1,785 bp gene in which the 285th amino acid was replaced by isoleucine to methionine. After designing and heat-treating at 94 ° C for 2 minutes, the DNA fragment of interest is obtained by PCR using a total of 35 cycles of 94 ° C for 15 seconds, 50 ° C for 30 seconds, and 68 ° C for 2 minutes. Amplified. The 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, and one end was further digested with SacI. Then, only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene). When E. coli JM109 was transformed with this DNA and the expression of the protein in the transformant was confirmed, it was confirmed that a recombinant protein having an expected molecular weight of about 75 kDa was expressed (FIG. 5, lane 2). ). The sequence of 595 amino acids encoding this protein is shown in SEQ ID NO: 3 described later.

  The protein of about 75 kDa (referred to as t96-7 DNA polymerase) was purified, and the DNA polymerase activity was examined by the RCA (Rolling Circle Amplification) method. A circular single-stranded DNA derived from Escherichia coli phage was used as a template, and primers were added to this DNA to verify the presence or absence of DNA polymerase activity and strand displacement activity. As the enzyme control, 5'-3 'exonuclease-deficient DNA polymerase derived from Geobacillus stearothermophilus was used (Bst DNA Polymerase, Large Fragment; New England Biolabs).

Specifically, M13mp18 single strand DNA 20 ng, Universal primer (5'-GTT TTC CCA GTC ACG ACG TTG TA-3 ') 50 nM, 0.25 mM each dNTPs, 20 mM Tris-HCl (pH 8.8 at 25 ° C) , 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Tween 20, and DNA polymerase 20 μl in total were reacted at 60 ° C for 0 to 30 minutes, followed by agarose gel electrophoresis As a result, the DNA band shifted to the polymer side depending on the reaction time, suggesting that t96-7 DNA polymerase has a strand displacement type DNA polymerase activity (FIG. 7).

Example 6
[Purification of 96-7-derived recombinant DNA polymerase]
The E. coli obtained in Example 5 is cultured and collected. E. coli is suspended in Sonication Buffer (200 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol, 0.1 mM Benzamidine, 10 μg / ml Bacitracin, 30 μg / ml Phenylmethylsulfonyl fluoride, 20 mM Tris-HCl (pH 7.5 at 25 ° C.)) Decompose genomic DNA by sonication while lowering the temperature on ice. The suspension is heated in a 55 ° C. water bath for 20 minutes. Immediately lower the temperature on ice, centrifuge and collect the supernatant.

  The supernatant was added to an anion exchange column (HiTrap Q HP; manufactured by GE Healthcare Bioscience), washed thoroughly, and separated by a linear NaCl gradient.

  RCA method and SDS-PAGE were performed to confirm fractions containing DNA polymerase. The desired fractions were collected, added to a heparin affinity column (HiTrap Heparin HP; manufactured by GE Healthcare Bioscience), washed thoroughly, and then separated by a NaCl linear concentration gradient.

  SDS-PAGE was performed to confirm the fraction containing DNA polymerase (FIG. 5, lane 3). Collected fractions were tested using well dialyzed against storage buffer (0.1 mM EDTA, 1 mM Dithiothreitol, 10 mM Tris-HCl (pH 7.5 at 25 ° C), 0.1% Tween 20, 50% Glycerol). The analysis in Example 1 was performed.

Test example 1
[Verification of reaction characteristics]

  The performance was verified in detail for tYMO81 DNA polymerase and t96-7 DNA polymerase from which the 5'-3 'exonuclease domain was removed.

Test Example 1-1
[Evaluation of optimum reaction temperature by RCA method]
The optimum reaction temperature of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by the RCA method. That is, the RCA method was performed at each temperature of 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, and 75 ° C. After reacting for 30 minutes, agarose gel electrophoresis was performed, and it was confirmed that the DNA band was shifted to the polymer side (FIG. 8). For each reaction, 4 units of Bst DNA Polymerase and Large Fragment, 400 ng of fYMO81 DNA polymerase, tYMO81 DNA polymerase, and t96-7 DNA polymerase were used.

  The strength of DNA polymerase activity and strand displacement activity was evaluated by the mobility of the band shift to the polymer side within a certain time. Bst DNA Polymerase and Large Fragment showed DNA polymerase activity in the temperature range of 50 to 65 ° C. In contrast, tYMO81 DNA polymerase had the maximum activity at 60 to 75 ° C, and t96-7 DNA polymerase had the maximum activity at 45 to 55 ° C. In addition, fYMO81 DNA polymerase also showed high molecular weight DNA suggesting strand displacement DNA polymerase activity, but formation of a large amount of small nucleic acid, probably due to endogenous 5'-3 'exonuclease activity. As a result, subsequent analysis on the reaction characteristics was not performed.

Test Example 1-2
[Evaluation of optimum reaction temperature by LAMP method]
The optimal reaction temperature of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by real-time turbidity measurement using the LAMP method. Specifically, positive control plasmid DNA, LAMP primer set, 0.25 mM each dNTPs, 20 mM Tris-HC (pH 8.8 at 25 ° C.), 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Tween 20, and a total of 25 μl of DNA polymerase were added to the Loopamp Real-time Turbidimeter LA-200 (manufactured by Teramex for 1 hour, and the increase in turbidity was observed. Real-time turbidity using the LAMP method In the degree measurement, the reaction time (Tt) required until the turbidity reached 0.1 was compared, and the lower the Tt value, the more efficiently the reaction progresses.

  The positive control plasmid DNA used here contains sequences characteristic of Candidatus Liberibacter asiaticus, a causative microorganism of citrus greening disease, or tobacco whitefly biotype Q, a vector insect of plant disease virus. The one designed for the purpose of specifically detecting the sequence of was used. As a LAMP primer set for specifically detecting Candidatus Liberibacter asiaticus, SEQ ID NO: 13 for FIP, SEQ ID NO: 14 for BIP, SEQ ID NO: 15 for F3, SEQ ID NO: 16 for B3, SEQ ID NO: 17 for LF, SEQ ID NO: 17 for LB A combination using 18 each primer was used. Further, as a LAMP primer set for specifically detecting tobacco whitefly biotype Q, a combination using SEQ ID NO: 19 for FIP, SEQ ID NO: 20 for BIP, SEQ ID NO: 21 for F3, and SEQ ID NO: 22 for B3 Using.

  For tYMO81 DNA polymerase, real-time turbidity measurement was performed at 60 ° C., 65 ° C., and 70 ° C. using a LAMP primer set and a positive control plasmid DNA for specifically detecting Candidatus Liberibacter asiaticus. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 800 ng of tYMO81 DNA polymerase were used.

  As a result, Bst DNA Polymerase and Large Fragment showed significantly higher Tt at 70 ° C, whereas tYMO81 DNA polymerase maintained low Tt even at 70 ° C. This suggests that it is possible to design a LAMP primer at a higher temperature than in the past (Table 1).

  For t96-7 DNA polymerase, real-time turbidity measurement was performed at 55 ° C. and 60 ° C. using a LAMP primer set for positive detection of tobacco whitefly biotype Q and a positive control plasmid DNA. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 600 ng of t96-7 DNA polymerase were used.

  Bst DNA Polymerase, Large Fragment has a significantly lower Tt at 55 ° C, while t96-7 DNA polymerase maintains a lower Tt at 55 ° C, enabling the design of LAMP primers at lower temperatures than before. (Table 2).

Test Example 1-3
[Evaluation of heat resistance of tYMO81 DNA polymerase]
Since tYMO81 DNA polymerase was suggested to have higher heat resistance than Bst DNA Polymerase, Large Fragment, tYMO81 DNA polymerase and Bst DNA for 5 minutes in advance at 70 ° C, 75 ° C, 80 ° C, 85, 90 ° C. After treating Polymerase and Large Fragment, real-time turbidity measurement using LAMP method was performed to evaluate whether the activity was maintained. Real-time turbidity measurement was performed at 65 ° C for 1 hour using a LAMP primer set and a positive control plasmid DNA for specifically detecting Candidatus Liberibacter asiaticus. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 800 ng of tYMO81 DNA polymerase were used.

  With Bst DNA Polymerase, Large Fragment, the increase in turbidity in the LAMP method was no longer observed after treatment at 70 ° C for 5 minutes, so it is expected that the protein activity would be significantly reduced when the treatment was performed at a high temperature of 70 ° C or higher. In contrast, tYMO81 DNA polymerase maintained activity even after treatment at 80 ° C. for 5 minutes, and the heat resistance of the protein was significantly improved (Table 3).

[Evaluation of heat resistance of t96-7 DNA polymerase]
Since t96-7 DNA polymerase was suggested to have higher heat resistance than Bst DNA Polymerase, Large Fragment, t96-7 in advance at each temperature of 65, 70 ° C, 75 ° C, 80 ° C, 85, 90 ° C, t96 -7 After treating DNA polymerase, Bst DNA Polymerase, and Large Fragment, real-time turbidity measurement was performed using the LAMP method to evaluate whether the activity was maintained. Real-time turbidity measurement was performed at 60 ° C. for 1 hour using a LAMP primer set and a positive control plasmid DNA for specifically detecting tobacco whitefly biotype Q. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 600 ng of t96-7 DNA polymerase were used.

  With Bst DNA Polymerase, Large Fragment, the increase in turbidity in the LAMP method was no longer observed after treatment at 70 ° C for 5 minutes, so it is expected that the protein activity would be significantly reduced when the treatment was performed at a high temperature of 70 ° C or higher. In contrast, with t96-7 DNA polymerase, no increase in turbidity was observed after 5 minutes of treatment at 65 ° C, but when t96-7 DNA polymerase without heat treatment was used, 60 ° C for 1 hour. Real-time turbidity measurement is possible, and it was suggested that the activity was maintained at least at 55-60 ° C, consistent with the results of maintaining the activity at lower temperatures than Bst DNA Polymerase and Lage Fragment ( Table 4).

Test Example 1-4
[Evaluation of optimum reaction pH by LAMP method]
The optimal reaction pH of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by real-time turbidity measurement using the LAMP method. Specifically, the change in the Tt value was observed when the pH of Tris-HCl contained in the LAMP method was changed from the original 8.8 up and down. The pH of Tris-HCl was adjusted under the temperature condition of 25 ° C. (Table 5).

  tYMO81 DNA polymerase was compared with Bst DNA Polymerase and Large Fragment using LAMP primer set and positive control plasmid DNA for specific detection of Candidatus Liberibacter asiaticus. That is, real-time turbidity measurement was performed at 65 ° C. for 1 hour at each pH of 7.5, 8.0, 8.3, 8.8, 9.0, 9.5, and 10.0. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 800 ng of tYMO81 DNA polymerase were used.

  Bst DNA Polymerase and Large Fragment had the lowest Tt at a pH around 8.3, whereas tYMO81 DNA polymerase had the lowest Tt in a wide pH range from 8.8 to 10.0. In general DNA synthesis, the pH of the reaction solution tends to be acidic due to the generation of reaction by-products and the temperature dependence of the Tris buffer, and the solubility of the protein may significantly reduce the DNA polymerase activity. As is known, tYMO81 DNA polymerase is expected to solve this problem by using a high pH buffer solution in advance, and to perform a stable reaction while maintaining the activity of DNA polymerase. .

  t96-7 DNA polymerase was compared with Bst DNA Polymerase, Large Fragment using LAMP primer set and positive control plasmid DNA for specifically detecting tobacco whitefly biotype Q. That is, real-time turbidity measurement was performed at 60 ° C. for 1 hour at each pH of 7.5, 8.0, 8.3, 8.8, and 9.0. For each reaction, 8 units of Bst DNA Polymerase and Large Fragment and 600 ng of t96-7 DNA polymerase were used.

  Bst DNA Polymerase and Large Fragment had the lowest Tt at pH around 8.3, whereas t96-7 DNA polymerase had the lowest Tt in the pH range from 8.3 to 9.0. Although t96-7 DNA polymerase does not reach tYMO81 DNA polymerase, it is expected to be usable on the basic side and in a wider range of pH conditions than Bst DNA Polymerase, Large Fragment.

Test Example 1-5
[Evaluation of base resistance by LAMP method]
In genetic diagnosis methods such as the LAMP method, the target nucleic acid to be amplified is extracted from a biological sample. In this process, a method of denaturing cellular proteins using a strongly basic solution such as sodium hydroxide is known. It has been. However, the remaining of the basic solution unintentionally increases the pH of the reaction solution and causes hydrolysis of protein molecules such as DNA polymerase. As a result, the DNA synthesis reaction by DNA polymerase is inhibited, so nucleic acid molecules are extracted from biological samples. In some cases, in order to remove these inhibitory substances, it is necessary to purify the nucleic acid by extraction with an organic solvent, concentration using a centrifugation operation, or separation. Therefore, the present inventors focused on the fact that the optimum pH of tYMO81 DNA polymerase is around 8.8 to 10.0 and is relatively biased toward the basic side. The impact was evaluated (Table 6).

  Specifically, a protocol for extracting genomic DNA of Candidatus Liberibacter asiaticus infected with citrus leaves was used. In other words, extraction of nucleic acid from citrus leaf specimens with 250 mM sodium hydroxide, neutralization with 2.5 M acetic acid followed by isopropanol precipitation, modification of the method for concentrating and purifying nucleic acids, and extraction with a reduced amount of acetic acid Real-time turbidity measurement using the LAMP method was performed under the condition where the solution was added directly.

  Usually, citrus leaves are placed in 250 μl of 250 mM sodium hydroxide and boiled, and then neutralized by adding 50 μl of 2.5 M acetic acid. The inventors prepared a mixed solution of sodium hydroxide and acetic acid in a pseudo manner, and positively added a LAMP primer set for specifically detecting Candidatus Liberibacter asiaticus under the condition that 2 μl thereof was added to the reaction solution. Real-time turbidity measurement was performed using control plasmid DNA. As a result, when the above mixed solution stock solution was used, no increase in turbidity was observed for both tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment. This is presumably because the ionic strength and pH of the solution greatly fluctuate due to high concentrations of sodium hydroxide or acetic acid, and the DNA polymerase reaction is inhibited.

  Therefore, the present inventors added the above mixed solution (referred to as a diluted extraction solution) after diluting it twice with sterilized distilled water and adding it, both tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment have turbidity. We found that there was an increase, and fixed the total volume of this diluted extract solution to 600 μl, the addition amount of 250 mM sodium hydroxide to 250 μl, and the acetic acid addition solution volume to 50 μl, 25 μl, 12.5 μl, 5 μl, 0 μl Real-time turbidity measurement was performed at 65 ° C for 1 hour using a LAMP primer set and a positive control plasmid DNA for specific detection of Candidatus Liberibacter asiaticus . Note that 8 units of Bst DNA Polymerase and Large Fragment and 500 ng of tYMO81 DNA polymerase were used per reaction. In addition, as a control for the diluted extract solution, a reaction in which only sterile distilled water was added was added instead.

  In Bst DNA Polymerase, Large Fragment, Tt increased markedly when the amount of acetic acid added was 5 μl or 0 μl, suggesting that neutralization of the basic solution was essential, whereas tYMO81 DNA polymerase On the contrary, Tt was the lowest when the added amount of acetic acid was 5 μl or around 0 μl. Therefore, when tYMO81 DNA polymerase is used, a nucleic acid extraction sample using a strong base solution can be directly input into a nucleic acid amplification reaction by omitting operations such as neutralization, concentration and solution removal. In the field of testing and diagnosis, it is expected to simplify the testing method by applying to a wider range of applications and automation than the conventional DNA polymerase. Interestingly, when the amount of acetic acid added is 12.5 μl, 5 μl, or 0 μl, Tt is remarkably reduced as compared with the case where only sterile distilled water is added as a control. This is consistent with the trend of the data in the present invention in which the optimum pH of tYMO81 DNA polymerase is distributed over a wide range on the basic side of 8.8 to 10.0, and tYMO81 DNA polymerase is inhibited by a strongly basic solution. Rather, it is believed to be activated and may inherently require basic reaction conditions.

Test Example 1-6
[Evaluation of protein storage stability by LAMP method]
In general genetic diagnosis and amplification of nucleic acids, the enzyme solution that is a component of the reaction is mostly a protein derived from Escherichia coli or any other microorganism, plant, or animal. Is often destabilized by factors such as proteolytic enzymes, temperature changes, and oxidation. In the field of genetic analysis, testing and diagnosis, genetic diagnostic methods such as the LAMP method must be excellent in reproducibility, and how stable the quality of DNA polymerase can be ignored is an important property. Accordingly, the present inventors focused on the fact that tYMO81 DNA polymerase does not lose its activity even at around 80 ° C., and compared the storage stability of tYMO81 DNA polymerase with Bst DNA Polymerase, Large Fragment. Specifically, no changes were observed in tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment in 24 hours during storage at 37 ° C. Therefore, under the conditions that increased the temperature load level, for example, the LAMP method is generally used here. Evaluation was made on the variation in residual activity at a temperature of 65 ° C., which is commonly used (Table 7). In Table 7, all were the average values of quadruple measurements.

  Bst DNA Polymerase, Large Fragment was evaluated for storage stability under conditions containing a commercially available storage solution, ie, buffer, salt, glycerol, and surfactant. It should be noted that tYMO81 DNA polymerase does not show a significant change in activity by 24 hours even when stored at 65 ° C under conditions including buffer, salt, glycerol, and surfactant. Increased the loading level and evaluated the storage stability in tYMO81 sterilized distilled water. The enzyme solution was stored at 65 ° C. for 3 hours, 6 hours, and 24 hours, and then evaluated using a LAMP primer set and a positive control plasmid DNA for specifically detecting Candidatus Liberibacter asiaticus. Note that 8 units of Bst DNA Polymerase and Large Fragment and 500 ng of tYMO81 DNA polymerase were used per reaction.

  In Bst DNA Polymerase, Large Fragment, a significant increase in Tt was confirmed by treatment at 65 ° C for 3 hours, and then Tt increased gradually after each of 6 hours and 24 hours. The decrease in activity at 3 hours is expected to be large. In contrast, with tYMO81 DNA polymerase, the Tt fluctuation is very small even after treatment at 65 ° C for 3 hours, the activity declines slowly after 6 hours, and no decline in activity is seen until 24 hours thereafter. It was. Usually, protein solutions are in the presence of protein stabilizers known as glycerol, bovine serum albumin, surfactants, protease inhibitors, etc., at a low temperature of about 2-8 ° C, or even at a low temperature range of -20 ° C. The tYMO81 DNA polymerase, which was stored as a nearly single protein molecule in sterile distilled water at about -80 ° C, did not lose most of its activity even at 65 ° C. It suggests long-term storage stability and resistance to loading conditions, and is a useful property.

The present invention can be used in any field where a strand displacement type DNA polymerase is used.

Claims (23)

A strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2. A temperature comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, having a strand displacement type DNA polymerase activity, and having a maximum strand displacement type DNA polymerase activity by the RCA method. A strand displacement DNA polymerase in the range of 60-75 ° C. It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has a strand displacement type DNA polymerase activity, and has a strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 70 ° C. A strand displacement DNA polymerase. A strand displacement type comprising an amino acid sequence having 90% or more identity with the amino acid sequence described in SEQ ID NO: 1 or 2, having strand displacement type DNA polymerase activity, and after heat treatment at a temperature of 70 to 80 ° C. for 5 minutes A strand displacement type DNA polymerase having a heat resistance of 80% or more of the strand displacement type DNA polymerase activity by the LAMP method when the DNA polymerase activity is not heat-treated. The strand displacement DNA polymerase according to claim 4, wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 60 to 75 ° C. The strand displacement type DNA polymerase according to claim 4, which has a strand displacement type DNA polymerase activity by a LAMP method at a temperature of 70 ° C. The strand displacement DNA polymerase according to any one of claims 2 to 6, wherein the pH at which the strand displacement DNA polymerase activity is maximized is 8.0 or more. The amino acid sequence shown in SEQ ID NO: 1 or 2 includes at least one or more biologically acceptable amino acid substitutions, amino acid deletions, and / or amino acid insertions. Strand displacement DNA polymerase. The strand displacement DNA polymerase according to claim 8, comprising 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions. A strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 3. It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has a strand displacement type DNA polymerase activity, and has a temperature at which the strand displacement type DNA polymerase activity by the RCA method becomes maximum at 45 to 45%. Strand displacement DNA polymerase in the range of 55 ° C. It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has strand displacement type DNA polymerase activity, and has strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 55 ° C. Strand displacement DNA polymerase. A strand-displacement DNA polymerase comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement-type DNA polymerase activity, and subjected to heat treatment at a temperature of 55-60 ° C for 5 minutes A strand displacement DNA polymerase having a heat resistance of 80% or more of the strand displacement DNA polymerase activity by the LAMP method when the activity is not heat-treated. The strand displacement DNA polymerase according to claim 13, wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 45 to 55 ° C. The strand displacement type DNA polymerase according to claim 13, which has strand displacement type DNA polymerase activity by the LAMP method at a temperature of 55 ° C. The strand displacement DNA polymerase according to any one of claims 11 to 15, wherein the pH at which the DNA polymerase activity and the strand displacement activity are maximized is 8.0 or more. The chain substitution according to any one of claims 11 to 16, wherein the amino acid sequence shown in SEQ ID NO: 3 comprises at least one biologically acceptable amino acid substitution, amino acid deletion, and / or amino acid insertion. DNA polymerase. The strand displacement DNA polymerase according to claim 17, comprising 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions. The strand displacement type DNA polymerase according to any one of claims 1 to 18, which is used for nucleic acid synthesis in a LAMP (Loop-mediated isothermal amplification) method. A gene encoding the DNA polymerase according to any one of claims 1 to 18. The gene according to claim 20 is mounted on a vector, a host cell is transformed with the vector, the transformed host cell is cultured, and the DNA polymerase encoded by the gene is accumulated in the culture, A method for producing a strand displacement type DNA polymerase, comprising collecting accumulated DNA polymerase. The production method according to claim 20, wherein the collected DNA polymerase comprises the strand displacement type DNA polymerase according to any one of claims 1 to 18. The production method according to claim 20 or 21, wherein an E. coli expression vector using a co-expression method with a leader sequence is used.